负重爬梯与有氧跑台运动对糖尿病大鼠学习记忆能力的影响及其机制探讨

目的：研究负重爬梯与有氧跑台运动对糖尿病大鼠学习记忆能力的改善效果并探索其可能分子机制。方法：40只雄性大鼠,随机分为正常对照组（NC）、糖尿病对照组（DC）、糖尿病负重爬梯组（DL）和糖尿病有氧跑台组（DA）,以单次腹腔注射链脲佐菌素构建糖尿病大鼠模型。DL组在晚上进行负重爬梯训练,10次/组×3组/天,每次间歇2 min,6天/周×6周;DA组在同一时间进行20 m/min的跑台训练,30 min/d。于造模成功和运动干预结束后采用Morris水迷宫检测大鼠的学习记忆能力;第2次水迷宫测试结束后断颈处死大鼠,采用RT-QPCR法检测大鼠海马内脑源性神经营养因子（BDNF）、TRKB、CREB mRNA表达水平。结果：与NC组相比,DC组大鼠海马BDNF、CREB基因表达显著下降,学习记忆能力显著降低。与DC组相比,DL和DA组大鼠海马BDNF、CREB基因表达显著上调,学习能力显著提高;DL大鼠海马TrkB基因显著上调,大鼠空间记忆能力显著改善,而DA组大鼠海马TrkB基因无显著变化,大鼠空间记忆能力无改善,与DA组相比,DL组大鼠海马TRKB、CREB基因显著上调。结论：有氧跑台运动与负重爬梯运动介导BDNF/TrkB/CREB信号通路对糖尿病大鼠的学习能力均有促进作用,而负重爬梯运动对糖尿病大鼠记忆能力的改善优于有氧运动方式。     

Alpha1-adrenergic receptor antagonist tamsulosin ameliorates aging-induced memory impairment by enhancing neurogenesis and suppressing apoptosis in the hippocampus of old-aged rats

Age-related memory decline is closely associated with decreased neurogenesis and increased apoptosis in the hippocampus. Noradrenaline exerts its effect by selectively binding to and activating adrenergic receptors (ARs). Tamsulosin, α₁-AR antagonist, is reported to have access to the brain and interact with α₁-AR. In this study, the effects of tamsulosin on short-term and spatial learning memory in terms of neurogenesis and apoptosis were investigated using rats. Step-down avoidance test for short-term memory and radial 8-arm maze test for spatial learning memory were conducted. Neurogenesis was detected by 5-bromo-2’-deoxyuridine (BrdU) immunohistochemistry and apoptosis was evaluated by caspase-3 immunohistochemisty and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNE) staining. Western blot for protein kinase C (PKC), cAMP-responsive element-binding protein (CREB), brain-derived neurotrophic factor (BDNF), tyrosine kinase B (TrkB), phosphatidylinositol 3-kinase (PI 3-kinase), Akt, Bcl-2, and Bax was conducted. In the aged rats, short-term and spatial learning memory was declined. Hippocampal nerogenesis was suppressed and hippocampal apoptosis was enhanced in the aged rats. In addition, phosphorylation of PKCα, CREB, PI-3 kinase, and Akt was decreased in the hippocampus of old-aged rats. Tamsulosin activated PKC/CREB and PI-3 kinase/Akt pathways. With these pathways, BDNF-TrkB signaling enhanced hippocampal neurogenesis and suppressed apoptosis in the old-aged rats. As the results, tamsulosin improved performance of short-term and spatial learning memory in the aged rats.     

Effects of Sevoflurane on Young Male Adult C57BL/6 Mice Spatial Cognition

Inhalation anesthetics are reported to affect cognition in both animals and humans. The influence of inhalation anesthetics in learning and memory are contradictory. We therefore investigated the effects of sevoflurane anesthesia with different durations on cognitive performance and the levels of NMDA receptor subunit NR2B, phosphorylated ERK1/2 (p-ERK1/2) and activated caspase3 in mouse hippocampus. We anaesthetized eight-week old male C57BL/6 mice with 2.5% sevoflurane for durations ranging from one to four hours. Non-anaesthetized mice served as controls. Mice exposed to sevoflurane for one to three hours showed improved performance, whereas mice with exposure up to four hours displayed similar behavioral performance as control group. NR2B was increased both at 24h and at two weeks post sevoflurane exposure in all groups. The p-ERK1/2: total ERK1/2 ratio increased at 24h in all anesthesia groups. The ratio remained elevated at two weeks in groups with two- to four-hour exposure. Activated caspase3 was detected elevated at 24h in groups with two- to four-hour exposure. The elevated trend of activated caspase3 was still detectable at two weeks in groups with three- to four-hour exposure. At two weeks post anesthesia, the typical morphology associated with apoptotic cells was observed in the hippocampus of mice exposed to four hours of sevoflurane. Our results indicate that 2.5% sevoflurane exposure for one to three hours improved spatial cognitive performance in young adult mice. The cognitive improvement might be related to the increase of NR2B, the p-ERK1/2: total ERK1/2 ratio in hippocampus. However, exposure to sevoflurane for four hours caused neurotoxicity due to caspase3 activation and apoptosis.     

A novel trimeric peptide, Neuropep-1-stimulating brain-derived neurotrophic factor expression in rat brain improves spatial learning and memory as measured by the Y-maze and Morris water maze

Abundant studies have shown possible links between low levels of brain-derived neurotrophic factor (BDNF) and neurological diseases such as Alzheimer's disease, Parkinson's disease, and depression, as well as stress and anxiety; therefore, BDNF could be a therapeutic target for neurological disorders. In the present study, a positional scanning-synthetic peptide combinatorial library was utilized to identify a peptide modulator of BDNF expression in the hippocampal neuronal cell line, H19-7. A novel tripeptide (Neuropep-1) induced a significant increase of BDNF mRNA and protein levels in H19-7 cells. Pre-treatment of TrkB inhibitor (K252a) did not block Neuropep-1-induced BDNF up-regulation. These results indicate that Neuropep-1 may up-regulate BDNF expression that might be independent of the TrkB receptor pathway. Tail vein injection of Neuropep-1 significantly up-regulated BDNF expression, TrkB phosphorylation, and its downstream signals including activation of Akt, ERK, and cAMP response element binding in the rat hippocampus. To evaluate improvement of spatial learning and memory (SLM) by Neuropep-1-induced BDNF up-regulation, the Y-maze and Morris water maze tests were performed. These results showed Neuropep-1 injection improved SLM performance with increase of BDNF and TrkB expression, activation of TrkB downstream signals in rat hippocampus compared with the control group. However, phosphorylation levels of TrkB were not changed when it was normalized to the level of TrkB expression. The difference on TrkB phosphorylation in Neuropep-1-injected rats may be affected by behavioral tests. These results suggest that Neuropep-1 may improve SLM via activation of the BDNF/TrkB signaling pathway in the rat hippocampus. Therefore, our findings represent that Neuropep-1 might be a potential candidate for treatment of learning and memory disorders as well as neurological diseases involving the abnormal expression of BDNF.     

Tau inhibits PKA by nuclear proteasome‐dependent PKAR2α elevation with suppressed CREB/GluA1 phosphorylation

Intraneuronal accumulation of wild‐type tau plays a key role in Alzheimer's disease, while the mechanisms underlying tauopathy and memory impairment remain unclear. Here, we report that overexpressing full‐length wild‐type human tau (hTau) in mouse hippocampus induces learning and memory deficits with remarkably reduced levels of multiple synapse‐ and memory‐associated proteins. Overexpressing hTau inhibits the activity of protein kinase A (PKA) and decreases the phosphorylation level of cAMP‐response element binding protein (CREB), GluA1, and TrkB with reduced BDNF mRNA and protein levels both in vitro and in vivo. Simultaneously, overexpressing hTau increased PKAR2α (an inhibitory subunit of PKA) in nuclear fraction and inactivated proteasome activity. With an increased association of PKAR2α with PA28γ (a nuclear proteasome activator), the formation of PA28γ‐20S proteasome complex remarkably decreased in the nuclear fraction, followed by a reduced interaction of PKAR2α with 20S proteasome. Both downregulating PKAR2α by shRNA and upregulating proteasome by expressing PA28γ rescued hTau‐induced PKA inhibition and CREB dephosphorylation, and upregulating PKA improved hTau‐induced cognitive deficits in mice. Together, these data reveal that intracellular tau accumulation induces synapse and memory impairments by inhibiting PKA/CREB/BDNF/TrkB and PKA/GluA1 signaling, and deficit of PA28γ‐20S proteasome complex formation contributes to PKAR2α elevation and PKA inhibition.     

Deoxygedunin对D-半乳糖联合AlCl3诱导大鼠阿尔茨海默病病理性改变的影响

目的：观察Deoxygedunin对D-半乳糖联合AlCl₃诱导的阿尔茨海默病模型大鼠Aβ沉积、学习记忆和氧化应激的影响及其可能机制。方法：健康成年雄性SD大鼠随机分为3组（n=12）：对照组（Control）、模型组（AD）和干预组（AD+Deo）。Morris水迷宫实验检测大鼠学习记忆和认知功能;采用酶联免疫吸附法（ELISA）检测大鼠海马组织匀浆中谷胱甘肽过氧化物酶（GSH-Px）、超氧化物歧化酶（SOD）和丙二醛（MDA）含量;免疫组织化学检测大鼠大脑皮层tau蛋白表达情况;Western blot实验用于检测TrkB信号通路上ERK1、AKT和TrkB蛋白的表达。结果：水迷宫结果显示,与对照组相比,D-半乳糖联合AlCl₃诱导的大鼠逃避潜伏期显著增加（P<0.05）。Deoxygedunin可逆转模型组逃避潜伏期的增加（P<0.05）。在去除平台的第7日,与对照组和干预组相比,模型组大鼠表现出逃避潜伏期增加（P<0.01）,穿越平台次数较少（P<0.05）;免疫组织化学和ELISA实验结果显示,与对照组相比,模型组Aβ、tau蛋白表达显著增加（P<0.01）,SOD、GSH-Px活性显著降低,MDA含量明显升高。与模型组相比,Deoxygedunin可逆转模型组Aβ、tau蛋白表达的增多（P<0.01）,SOD、GSH-Px活性显著降低（P<0.05）,MDA含量明显升高（P<0.05）;Western blot结果显示,Deoxygedunin干预可逆转模型组海马TrkB、AKT和ERK1的磷酸化水平的降低。结论：Deoxygedunin可通过激活TrkB信号转导通路显著逆转Aβ沉积,氧化应激和认知缺陷,提示Deoxygedunin可能作为减轻D-半乳糖联合AlCl₃诱导AD样病理功能障碍潜在的治疗备选药物。     

Genetic Enhancement of Memory and Long-Term Potentiation but Not CA1 Long-Term Depression in NR2B Transgenic Rats

One major theory in learning and memory posits that the NR2B gene is a universal genetic factor that acts as rate-limiting molecule in controlling the optimal NMDA receptor''s coincidence-detection property and subsequent learning and memory function across multiple animal species. If so, can memory function be enhanced via transgenic overexpression of NR2B in another species other than the previously reported mouse species? To examine these crucial issues, we generated transgenic rats in which NR2B is overexpressed in the cortex and hippocampus and investigated the role of NR2B gene in NMDA receptor-mediated synaptic plasticity and memory functions by combining electrophysiological technique with behavioral measurements. We found that overexpression of the NR2B subunit had no effect on CA1-LTD, but rather resulted in enhanced CA1-LTP and improved memory performances in novel object recognition test, spatial water maze, and delayed-to-nonmatch working memory test. Our slices recordings using NR2A- and NR2B-selective antagonists further demonstrate that the larger LTP in transgenic hippocampal slices was due to contribution from the increased NR2B-containing NMDARs. Therefore, our genetic experiments suggest that NR2B at CA1 synapses is not designated as a rate-limiting factor for the induction of long-term synaptic depression, but rather plays a crucial role in initiating the synaptic potentiation. Moreover, our studies provide strong evidence that the NR2B subunit represents a universal rate-limiting molecule for gating NMDA receptor''s optimal coincidence-detection property and for enhancing memory function in adulthood across multiple mammalian species.     

Neuroprotection of Cytisine Against Cerebral Ischemia–Reperfusion Injury in Mice by Regulating NR2B-ERK/CREB Signal Pathway

The aim of the study was to elucidate the therapeutic effects of Cytisine (CYT) on cerebral ischemia–reperfusion injury in mice. Male ICR mice were pretreated with reagents (drug), and then subjected to 2 h focal cerebral ischemia and 24 h reperfusion. Morphologically, the histopathological impairment were estimated by the TTC, HE and TUNEL staining. The expression of GluN2B-containing NMDA receptor, phosphorylation of extracellular regulated protein kinases, total ERK, phosphorylation of cAMP-response element binding protein and total CREB were determined by immunofluorescence and Western blot assay, respectively. The mRNA expression of NR2B, ERK and CREB were quantified by the real-time RT-PCR. CYT significantly diminished the infarct size and neuronal apoptosis. Additionally, it ameliorated histopathological lesion dramatically. CYT promoted the phosphorylation of ERK, CREB and their mRNA expression. In contrast, the expression of NR2B was suppressed in concomitant with the down-regulation of genes. The overall results thus far suggest that CYT confers the neuroprotection against cerebral I/R injury by regulating the NR2B-ERK/CREB signal pathway.     

NR2B-containing NMDA receptors promote neural progenitor cell proliferation through CaMKIV/CREB pathway

Accumulating evidence indicates the involvement of N-methyl-d-aspartate receptors (NMDARs) in regulating neural stem/progenitor cell (NSPC) proliferation. Functional properties of NMDARs can be markedly influenced by incorporating the regulatory subunit NR2B. Here, we aim to analyze the effect of NR2B-containing NMDARs on the proliferation of hippocampal NSPCs and to explore the mechanism responsible for this effect. NSPCs were shown to express NMDAR subunits NR1 and NR2B. The NR2B selective antagonist, Ro 25-6981, prevented the NMDA-induced increase in cell proliferation. Moreover, we demonstrated that the phosphorylation levels of calcium/calmodulin-dependent protein kinase IV (CaMKIV) and cAMP response element binding protein (CREB) were increased by NMDA treatment, whereas Ro 25-6981 decreased them. The role that NR2B-containing NMDARs plays in NSPC proliferation was abolished when CREB phosphorylation was attenuated by CaMKIV silencing. These results suggest that NR2B-containing NMDARs have a positive role in regulating NSPC proliferation, which may be mediated through CaMKIV phosphorylation and subsequent induction of CREB activation.     

Cell Signaling and Differential Protein Expression in Neuronal Differentiation of Bone Marrow Mesenchymal Stem Cells with Hypermethylated Salvador/Warts/Hippo (SWH) Pathway Genes

Human mesenchymal stem cells (MSCs) modified by targeting DNA hypermethylation of genes in the Salvador/Warts/Hippo pathway were induced to differentiate into neuronal cells in vitro. The differentiated cells secreted a significant level of brain-derived neurotrophy factor (BDNF) and the expression of BDNF receptor tyrosine receptor kinase B (TrkB) correlated well with the secretion of BDNF. In the differentiating cells, CREB was active after the binding of growth factors to induce phosphorylation of ERK in the MAPK/ERK pathway. Downstream of phosphorylated CREB led to the functional maturation of differentiated cells and secretion of BDNF, which contributed to the sustained expression of pERK and pCREB. In summary, both PI3K/Akt and MAPK/ERK signaling pathways play important roles in the neuronal differentiation of MSCs. The main function of the PI3K/Akt pathway is to maintain cell survival during neural differentiation; whereas the role of the MAPK/ERK pathway is probably to promote the maturation of differentiated MSCs. Further, cellular levels of protein kinase C epsilon type (PKC-ε) and kinesin heavy chain (KIF5B) increased with time of induction, whereas the level of NME/NM23 nucleoside diphosphate kinase 1 (Nm23-H1) decreased during the time course of differentiation. The correlation between PKC-ε and TrkB suggested that there is cross-talk between PKC-ε and the PI3K/Akt signaling pathway.     

Effect of ketamine on ERK expression in hippocampal neural cell and the ability of learning behavior in minor rats

To study the effects of ketamine on ERK expression in hippocampal neural cell and the ability of learning behavior in minor rats. Seventy-two Sprague–Dawley rats of 21 days old were randomly divided into nine groups. The Y-maze was used to test the ability of learning and spatial localization. At the end of training, all rats were killed and the expression levels of ERK1, ERK2 and p-ERK1/2 were tested by immunohistochemistry. The learning times and total reaction time (TRT) of group K2a, K2b, K2c and K3 have significant differences compared with T group (P < 0.05). Immunohistochemical staining showed that the level of ERK1, ERK2 and p-ERK1/2 in all rats which received light-electricity integrated training increased remarkably relative to the C group (P < 0.01). The expression levels of ERK1, ERK2 and p-ERK1/2 in hippocampal neural cell of group K2a, K2b and K3 significantly decreased when compared with T group (P < 0.05). Therefore, the results demonstrate that administration of over-anesthetic ketamine may impair learning ability of 21 days old rats within 24 h. ERK signal transduction pathway may be involved in the ability of learning and spatial localization. The inhibition of ERK signal transduction pathway may be one of the mechanisms of the impairment of learning and memory ability by ketamine.     

Negative regulation of neurogenesis and spatial memory by NR2B-containing NMDA receptors

Several lines of evidence suggest involvement of NMDA receptors (NMDARs) in the regulation of neurogenesis in adults and the formation of spatial memory. Functional properties of NMDARs are strongly influenced by the type of NR2 subunits incorporated. In adult forebrain regions such as the hippocampus and cortex, only NR2A and NR2B subunits are available to form the receptor complex with NR1 subunit. NR2B is predominant NR2 subunit in any of rat or human neural stem cells (NSCs). Thus, we suppose that NR2B-containing NMDAR should be critical in regulating adult neurogenesis, and thereby playing a role in the formation of spatial memory. In the cultured NSCs derived from the embryonic brain of rats, NR2B subunit-specific NMDAR antagonist Ro25-6981 increased cell proliferation, whereas MK-801, non-selective open-channel blocker of NMDARs, inhibited cell proliferation. Blockade of NR2B-containing NMDAR stimulated neurogenesis in the adult hippocampus and facilitated the formation of spatial memory. The enhanced spatial memory dropped back to base level when the NR2B antagonist-induced neurogenesis was neutralized by 3'-azido-deoxythymidine, a telomerase inhibitor. In addition, blockade of NR2B inhibited neuronal nitric oxide synthase (nNOS) enzymatic activity. In null mutant mice lacking nNOS gene (nNOS−/−), the effects of NR2B antagonist on neurogenesis disappeared. Moreover, nitric oxide donor DETA/NONOate attenuated and nNOS inhibitor 7-nitroindazole enhanced the effect of Ro 25-6981 on NSCs proliferation. Our findings suggest that NR2B-containing NMDAR subtypes negatively regulate neurogenesis in the adult hippocampus by activating nNOS activity and thereby hinder the formation of spatial memory.     

Efficacy of Lovastatin on Learning and Memory Deficits Caused by Chronic Intermittent Hypoxia-Hypercapnia: Through Regulation of NR2B-Containing NMDA Receptor-ERK Pathway

Background

Chronic intermittent hypoxia-hypercapnia (CIHH) exposure leads to learnning and memory deficits in rats. Overactivation of N-methyl-D-aspartate receptors(NMDARs) can lead to the death of neurons through a process termed excitotoxicity, which is involved in CIHH-induced cognitive deficits. Excessively activated NR2B (GluN2B)-containing NMDARs was reported as the main cause of excitotoxicity. The ERK1/2 (extracellular signal-regulated kinase 1/2) signaling cascade acts as a key component in NMDARs-dependent neuronal plasticity and survival. Ca2+/calmodulin-dependent protein kinase II (CaMKII), synapse-associated protein 102 (SAP102) and Ras GTPase-activating protein (SynGAP) have been shown to be involved in the regulation of NMDAR-ERK signalling cascade. Recent studies revealed statins (the HMG-CoA reductase inhibitor) have effect on the expression of NMDARs. The present study intends to explore the potential effect of lovastatin on CIHH-induced cognitive deficits and the NR2B-ERK signaling pathway.

Methods and Findings

Eighty male Sprague Dawley rats were randomly divided into five groups. Except for those in the control group, the rats were exposed to chronic intermittent hypoxia-hypercapnia (CIHH) (9∼11%O₂, 5.5∼6.5%CO₂) for 4 weeks. After lovastatin administration, the rats performed better in the Morris water maze test. Electron microscopy showed alleviated hippocampal neuronal synaptic damage. Further observation suggested that either lovastatin or ifenprodil (a selective NR2B antagonist) administration similarly downregulated NR2B subunit expression leading to a suppression of CaMKII/SAP102/SynGAP signaling cascade, which in turn enhanced the phosphorylation of ERK1/2. The phosphorylated ERK1/2 induced signaling cascade involving cAMP-response element-binding protein (CREB) phosphorylation and brain-derived neurotrophic factor (BDNF) activation, which is responsible for neuroprotection.

Conclusions

These findings suggest that the ameliorative cognitive deficits caused by lovastatin are due to the downregulation of excessive NR2B expression accompanied by increased expression of ERK signaling cascade. The effect of NR2B in upregulating pERK1/2 maybe due, at least in part, to inactivation of CaMKII/SAP102/SynGAP signaling cascade.     

Blueberry-induced changes in spatial working memory correlate with changes in hippocampal CREB phosphorylation and brain-derived neurotrophic factor (BDNF) levels

Phytochemical-rich foods have been shown to be effective at reversing age-related deficits in memory in both animals and humans. We show that a supplementation with a blueberry diet (2% w/w) for 12 weeks improves the performance of aged animals in spatial working memory tasks. This improvement emerged within 3 weeks and persisted for the remainder of the testing period. Memory performance correlated well with the activation of cAMP-response element-binding protein (CREB) and increases in both pro- and mature levels of brain-derived neurotrophic factor (BDNF) in the hippocampus. Changes in CREB and BDNF in aged and blueberry-supplemented animals were accompanied by increases in the phosphorylation state of extracellular signal-related kinase (ERK1/2), rather than that of calcium calmodulin kinase (CaMKII and CaMKIV) or protein kinase A. Furthermore, age and blueberry supplementation were linked to changes in the activation state of Akt, mTOR, and the levels of Arc/Arg3.1 in the hippocampus, suggesting that pathways involved in de novo protein synthesis may be involved. Although causal relationships cannot be made among supplementation, behavior, and biochemical parameters, the measurement of anthocyanins and flavanols in the brain following blueberry supplementation may indicate that changes in spatial working memory in aged animals are linked to the effects of flavonoids on the ERK-CREB-BDNF pathway.     

Activation of extracellular signal-regulated kinases, NF-kappa B, and cyclic adenosine 5'-monophosphate response element-binding protein in lung neutrophils occurs by differing mechanisms after hemorrhage or endotoxemia

Acute lung injury is frequently associated with sepsis or blood loss and is characterized by a proinflammatory response and infiltration of activated neutrophils into the lungs. Hemorrhage or endotoxemia result in activation of cAMP response element-binding protein (CREB) and NF-kappa B in lung neutrophils as well as increased expression of proinflammatory cytokines, such as TNF-alpha and macrophage-inflammatory peptide-2, by these cells. Activation of the extracellular regulated kinase (ERK) pathway occurs in stress responses and is involved in CREB activation. In the present experiments, hemorrhage or endotoxemia produced increased activation of mitogen-activated protein kinase kinase (MEK)1/2 and ERK2 (p42), but not of ERK1 (p44), in lung neutrophils. ERK1, ERK2, and MEK1/2 were not activated in peripheral blood neutrophils after hemorrhage or endotoxemia. Inhibition of xanthine oxidase led to further increase in the activation of MEK1/2 and ERK2 in lung neutrophils after hemorrhage, but not after endotoxemia. Alpha-adrenergic blockade before hemorrhage resulted in increased activation in lung neutrophils of MEK1/2, ERK1, ERK2, and CREB, but decreased activation of NF-kappa B. In contrast, alpha-adrenergic blockade before endotoxemia was associated with decreased activation of MEK1/2, ERK2, and CREB, but increased activation of NF-kappa B. Beta-adrenergic blockade before hemorrhage did not alter MEK1/2 or ERK1 activation in lung neutrophils, but decreased activation of ERK2 and CREB, while increasing activation of NF-kappa B. Beta-adrenergic inhibition before endotoxemia did not affect activation of MEK1/2, ERK1, ERK2, CREB, or NF-kappa B. These data indicate that the pathways leading to lung neutrophil activation after hemorrhage are different from those induced by endotoxemia.